Light sensing device

ABSTRACT

The present invention relates to a light scanning device for exciting and detecting an emission of secondary light, especially fluorescent light, of a sample, comprising a light generating device for generating scanning light in the form of a single light beam, a deflection unit used for effecting a deflection of the scanning light for scanning at least one subarea of the sample, said deflection being variable in at least one direction, an imaging unit for forming an image of the secondary light emanating from the sample, and a detection unit for detecting the secondary light. When a sample with a large surface to be rastered is subjected to fluorescence examination with high spatial resolution, undesirably long scanning times occur. For reducing the scanning time and for simultaneously maintaining the high resolution in the case of such a sample, the light scanning device according to the present invention comprises a division device for dividing the single light beam into at least two light beams. This has the effect that, instead of the former sequential scanning of the sample, a subdivision into fields is carried out, said fields being scanned simultaneously by the plurality of light beams. The scanning time can therefore be reduced in accordance with the number of the simultaneously scanned fields of the sample.

FIELD OF THE INVENTION

The present invention relates to a light scanning device for excitingand detecting an emission of secondary light, especially fluorescentlight, of a sample, comprising a light generating device for generatingscanning light in the form of a single light beam, a deflection unitused for effecting a deflection of the scanning light for scanning atleast one subarea of the sample, said deflection being variable in atleast one direction, an imaging unit for forming an image of thesecondary light emanating from the sample, and a detection unit fordetecting the secondary light.

BACKGROUND ART

Light scanning devices of the above-mentioned type are used e.g. for aspatially resolved fluorescence examination of a sample. For thispurpose, the above-mentioned device for generating the scanning light inthe form of a single light beam produces a narrow beam, which isfocussed onto the sample and which is rastered over the sample by meansof a deflection device, e.g. in the form of tilting mirrors with twoorthogonal tilting axes or axes of rotation in the optical path of thelight beam, said light-generating device being a laser in most cases.The scanning light excites on the surface of a sample the generation ofsecondary light, e.g. in the form of fluorescent light. This secondarylight is collected via an imaging optics and detected on a detectionunit. Since the deflection unit irradiates, in a precisely definablemanner, a respective specific spot on the sample in dependence upon theposition of the tilting mirrors relative to one another and relative tothe sample, a locally dependent statement with regard to the respectiveproperty of the sample can be made by means of the detection unitdetecting the intensity of the secondary light.

The scanning time for measuring the whole sample depends on variousparameters, such as the size of the angular field on the sample, thescanning increment, the spot size of the scanning beam on the sample,the integration time of the detection unit, the scanning or mirrorvelocity of the deflection unit as well as the desired signal-to-noiseratio. When samples with dimensions in the centimeter range are scannedwith high spatial resolution by a scanning beam focussed to a fewmicrometers, the scanning times are in the range of minutes to hours.Such long scanning times are, however, a great problem for the operationof light scanning devices of this kind.

SUMMARY OF THE INVENTION

It is therefore the object of the present invention to provide animproved light scanning device which can be used for scanning a sampleand for detecting secondary radiation excited by the scanning light andby means of which a faster and more efficient scanning of a large samplewith high spatial resolution can be accomplished.

According to the present invention, this object is achieved by a lightscanning device of the type cited at the start, which is characterizedin that a division device is provided for dividing the single light beaminto at least two light beams. Due to the division of the single lightbeam into at least two light beams, the whole surface of the sample isno longer scanned sequentially, as has hitherto been the case, but atleast two areas of the sample are rastered simultaneously by the atleast two scanning beams. Hence, the scanning time can essentially behalved, the spatial resolution remaining the same.

According to an advantageous further development of the presentinvention, the division device comprises at least one, preferably,however, two wedge-shaped bimirrors, especially beam splitters,comprising each a first and a second surface at which the singleincoming beam is reflected, whereby two beams are formed, said two beamsenclosing an angle which corresponds to the wedge angle of the beamsplitter. When two beam splitters are used in accordance with apreferred embodiment, four beams are produced from the initially singlebeam. Due to the division of the incoming beam into four beams, thesample is subdivided into four quadrants, whereby the scanning time canessentially be reduced to a quarter of the scanning time required when asingle beam is used. The two reflecting, wedge-shaped beam splitters orbeam splitters are, advantageously, a part of the deflection unit andrepresent the respective tilting mirrors with orthogonal axes ofrotation of this unit, said tilting mirrors being coupled with suitableadjusting elements.

In accordance with an additional advantageous further development of thepresent invention, a focussing lens is provided between the deflectionunit and the sample. It will especially be of advantage to use an F/Θlens which focusses the light beams sharply independently of thedisplacement, i.e. the distance from the optical axis. This kind ofarrangement of the focussing lens between the deflection unit and thesample is referred to as “pre-objective-scanning”.

According to an additional advantageous further development, thedetection unit consists of a spatially resolving detector array, e.g. aCCD camera or a multi-channel multiplier or a multi-channelsemiconductor element. For reducing undesired cross-talk between theindividual channels, which correspond to the areas on the sample scannedby the individual light beams, a special diaphragm, which is adapted tothe respective sample areas scanned, can be provided in front of thedetector.

In the case of measurements in a transmissive arrangement, it will beadvantageous to provide, if possible, the whole surface behind thesample with light guides, the light guides associated with each scanningarea of the sample being combined so as to form a bundle and beingconducted to a respective detector area or to a detector of their own.For example, if the sample is subdivided into four quadrants, the lightguides are combined so as to form four bundles and are conducted ontofour different detectors so that the four quadrants can be measuredsimultaneously. In this connection it is also possible to arrange colourfilters in front of the detectors for suppressing the excitation lighton the one hand and for carrying out a selection of the secondary lighton the other. The numerical aperture of the light guides restricts theangular field of secondary light emission and prevents thereforecross-talk between the channels. If the sample consists of fluorescentdyes of the same kind, each of the detectors associated with a scanningfield of the sample can be equipped with a different colour filter sothat, if e.g. four detectors are used, four different emissionwavelengths can be measured simultaneously.

Instead of using different detectors coupled to the sample vialight-guide bundles, it would also be possible to arrange, according toa further development of the present invention, a CCD camera behind thesample in a transmissive arrangement. For preventing the fluorescentlight of all channels from being mixed in the camera, a plate consistingof light-conducting fibres having a small numerical aperture is placedin front of the camera, whereby cross-talk between the channels can beprevented effectively.

According to an additional advantageous further development of the lightscanning device according to the present invention, a set-up is providedfor detecting the secondary light in a reflective, non-confocalarrangement. For creating said non-confocal arrangement, i.e. forimplementing the ray path of the secondary light in such a way that themirrors of the deflection unit are not included in the ray path of saidsecondary light, a dichroic beam splitter is advantageously providedbetween the deflection unit and the sample, said dichroic beam splitterbeing adapted to be used for separating the optical path of the scanninglight from the optical path of the secondary light emanating from thesample. Especially, the dichroic beam splitter transmits the excitationlight having a first shorter wavelength, whereas it reflects thesecondary light having a longer wavelength.

Further advantageous embodiments are disclosed by the sub-claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Making reference to the accompanying drawings, the present inventionwill be explained and described in more detail on the basis of apreferred embodiment serving as an example.

In the said drawings,

FIG. 1 shows a sketch for illustrating the fundamental set-up and theray path of a light scanning device according to the present invention,

FIG. 2 shows an example of the division device in the form of awedge-shaped beam splitter plate with a suitable ray path;

FIG. 3 shows a sketch for illustrating the various detection channels ina light scanning device according to the present invention; and

FIG. 4 shows a sketch for illustrating the subdivision of a sample intofour quadrants.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

FIG. 1 shows, by way of example, a schematic set-up of a light scanningdevice according to the present invention. In a light-generating device20, e.g. a laser, a single light beam 21 having a specific wavelength isproduced. The light beam produced by the light-generating device orrather the laser is spatially filtered in a spatial filter in anadvantageous manner, said spatial filter being not shown. The bundle ofparallel rays is expanded by means of an expansion optics comprisinge.g. two lenses 22 and 24, so as to form a beam having a largercross-section, said beam being again a bundle of parallel rays. The nextelement following in the optical path is a deflection unit 10 comprisingtilting mirrors 11 and 12 which have orthogonal axes of rotation andwhich are connected to adjusting elements, not shown, provided withsuitable control means for adjusting or tilting said tilting mirrorsrelative to one another so as to raster the beam 21 in two directions.As will be explained in detail hereinbelow with regard to FIG. 2, thetilting mirrors 11 and 12 each consist of reflecting, wedge-shaped beamsplitters or beam splitter plates in an advantageous embodiment of thepresent invention, said reflecting beam splitters or beam splitterplates defining consequently a division device for dividing the singleincoming laser beam into a total of four separate laser beams. Forreasons of clarity, only two beams 22 and 23 are shown in FIG. 1.

Between the deflection unit 10 comprising the tilting mirrors 11 and 12,a focussing optics is arranged, which comprises e.g. a triplet lensconsisting of the lenses 26, 28 and 30. This focussing optics consistsadvantageously of an F/Θ lens which, indpendently of the displacement,focusses the beam sharply to spot sizes in the micrometer range on asample 40. When an F/Θ lens is used, the scanning beams are imagedaccording to the so-called F/Θ condition y′=F/Θ, wherein y′ is theimaging coordinate, F the focal length and e the angle enclosed by thescanning beam and the optical axis. In contrast to conventional lenses,where the normally applicable condition y′=F×tan Θ holds true, the F/Θlens causes barrel distortion. This, however, guarantees aporportionality between the scanning angle and the image height y′ andsimultaneously also a proportionality between the angular velocity ofthe deflection system and the scanning velocity in the sample plane. Itfollows that, when the angular velocity for the deflection of the beamis constant, a constant excitation intensity on the sample will becreated, independently of the scanning position, due to the linearitybetween the scanning velocity on the sample and the angular velocity.

This kind of arrangement of the focussing optics between the deflectionunit 10 comprising the tilting mirrors 11 and 12 and the sample 40 isreferred to as “pre-objective scanning”. This is used more frequentlythan “post-objective scanning” where the focussing optics is arranged inthe optical path in front of the deflection unit 10 so that the scanninglight, which is convergent after the focussing optics, is deflected viathe deflection mirrors and directed onto the sample 40. In the case ofthis kind of arrangement of the focussing optics in front of thedeflection unit 10, the lens only has to fulfil minimal demands. It mayhave a small diameter and it only has to form sharp images in theparaxial region. The deflection unit arranged behind the lens results,however, in a curved scanning line located on a circular arc about theaxis of rotation of the tilting mirror. This “post-objective scanning”arrangement is therefore not preferred for scanning plane surfaces.

Hence, it will be advantgeous to use the “pre-objective scanning”arrangement comprising an F/Θ lens, which can be used for forming imagesin a plane with an image coordinate that is proportional to thedeflection angle. The F/e lens in the “pre-objective scanning”arrangement must, however, have a comparatively large diameter so thatit will also accept scanning beams having a large scanning angle. Itmust also be corrected over a comparatively large angular fieldaccording to the tilting of the light beam relative to the axis and, inaddition, it must have a good field flatness.

A dichroitic mirror 32 is arranged between the focussing optics and thesample 40, said dichroic mirror 32 permitting passage of the excitationlight having the specific wavelength and reflecting the secondary lightwhich is generated on or by the sample 40 and which has a wavelengththat is different from, i.e. longer than that of the excitation light.In the reflection direction of the dichroic mirror 32 a collectingoptics 34 and a detector 50 are arranged after said dichroic mirror.

The arrangement shown in FIG. 1 represents a case in which the secondarylight is measured in a reflective, non-confocal arrangement. Thenon-confocal arrangement has the advantage that a larger solid angle forthe secondary light emitted by the sample can be accepted than in thecase of confocal imaging.

Notwithstanding this, also a confocal arrangement (not shown in FIG. 1)would be possible in contrast thereto; in such a confocal arrangement,the secondary light emitted by the sample is guided back via the sametilting mirrors 11 and 12 so that the light travels along a ray pathwhich corresponds exactly to that of the scanning light but in theopposite direction. In this arrangement, the dichroic mirror would beprovided between the light source 20 and the deflection unit 10 so as toseparate the optical paths of the excitation light and of thewavelength-displaced secondary light. By additionally focussing thesecondary light onto a pinhole diaphragm (not shown), e.g. a pinholediaphragm having a hole in the micrometer range, undesired stray lightcould be suppressed to a large extent. The confocal arrangement,however, only accepts a very small solid angle of the secondary light,which is limited by the mirror apertures, when a comparatively largeangular field is to be scanned simultaneously.

In contrast to the arrangement shown in FIG. 1, it would also bepossible to measure in a transmissive arrangement instead of areflective arrangement. In a transmissive arrangement, the scanning andexcitation light, respectively, must be blocked with the aid of filters(not shown) (e.g. notch filters or cut-off filters), which, in turn,reflect light onto the sample and which therefore cause blurring of theexcitation light spot on the sample. Hence, the reflective arrangementshown in FIG. 1 is advantageous in comparison with the transmissivearrangement.

FIG. 2 shows in the form of an example how the division device fordividing the single light beam into at least two light beams can berealized according to the present invention. The division deviceconsists of a wedge-shaped beam splitter plate 110, the respective lightrays of an incident light beam 116 being reflected on the first surface112 and on the second surface 114 of said beam splitter plate 110 so asto produce two light beams 118 and 119. It will be advantageous when thetilting mirror 12 shown in FIG. 1 is implemented in the form of thewedge-shaped beam splitter plate 110 shown in FIG. 2. In particular, itwill be advantageous when each of the two tilting mirrors 11 and 12 ofthe deflection unit 10 in FIG. 1 is implemented in the form of a beamsplitter plate 110 of the kind shown in FIG. 2, whereby the singleincident light beam will be divided into four light beams. In this case,the sample 40 is subdivided into four quadrants 43, 44, 45 and 46 asshown in FIG. 4. The area of each quadrant of the sample is thereforescanned by one of the four beams of the scanning light by means ofadjusting the tilting mirrors in a suitable manner. It follows that,when a sample having macroscopic dimensions of approx. 24×24 mm isscanned, the time required for complete scanning of this sample will bereduced to a quarter of the hitherto necessary time. It is thereforepossible to focus the scanning light beams more strongly than hashitherto been the case so as to increase the spatial resolution in thescanning process, the time required for scanning a large sample beingstill acceptable.

When the scanning light beam is focussed onto a point, stray light canalso reach neighbouring molecules whose secondary light will, in thecase of non-focal imaging, be included in the measurement and associatedwith the instantaneous position of the tilting mirrors of the scanningunit. Notwithstanding this, a non-confocal set-up could still be ofadvantage, since, when a confocal set-up is used, the signal is muchsmaller due to the smaller solid angle. In addition, the above-mentioneddifficulty with regard to stray light is reduced by the arrangementaccording to the present invention, since an increase in the resolution,i.e. stronger focussing of the scanning beam without a resultantincreased expenditure of time for the scanning of the sample, ispossible.

A further advantage of the embodiment described is to be seen in thefact that, due to the division of the scanning light beam into fourlight beams and due to a corresponding division of the sample into fourquadrants, each of the scanning light beams only has to cover a smallerarea. Hence, only minor deflections of the tilting mirrors 11 and 12 arerequired, which can be realized with smaller errors and tolerances,respectively. The tilting movements of the tilting mirrors are adaptedin such a way that the quandrants 43-46 shown in FIG. 4 are each sweptcompletely by the respective sub-beams.

The wedge angle of the beam splitter plates is, in an advantageousmanner, large enough to make the macroscopic distance between theindividual beams on the sample large in comparison with the meanscattering length within the sample. In the case of an exemplary samplehaving an area of 24×24 mm, this distance has an optimum value of 12 mm.

FIG. 3 shows schematically the fundamental structural design of thevarious detection channels. Secondary light, which is shown in the formof beams 61 and 62, is generated at two points of the sample 40. As hasalready been mentioned, this light is reflected at the dichroic mirror32 and is then focussed onto the detector 50 through a macrolensconsisting e.g. of three lenses 35, 36 and 37. Each point of the sampleplane corresponds unequivocally to a point in the detector plane. Thedetector is advantageously a CCD camera or a multi-channelphotomultiplier, e.g. the model R5900U-00-M4 by Hamamatsu, or amulti-channel semiconductor element. Detectors of this kind are suitablefor simultaneously detecting a plurality of channels. The respectivescanning fields of the sample (e.g. four quadrants) are correspondinglyimaged on the detector plane. For suppressing an undesirable cross-talkbetween the channels, a special diaphragm 52, which is adapted to thesubdivision of the sample, can be provided in front of the detector 50.For the above-mentioned embodiment with four scanning light beams, thediaphragm 52 is subdivided into four quandrants in a correspondingmanner.

In contrast to the arrangement for measuring the secondary light inreflection, which is shown in FIG. 1 and 3, it is also possible tomeasure in a transmissive arrangement. In such a transmissivearrangement, e.g. the whole surface behind the sample is provided withlight guides, the light guides of each quadrant being combined so as toform one bundle. Four bundles are then conducted onto four differentdetectors which can be measured simultaneously. For suppressing theexcitation light and for selecting the secondary light, special filterscan be provided in front of the detectors. The numerical aperture of thelight guides restricts the angular field of secondary light emission andprevents therefore cross-talk between the channels. If the sampleconsists of fluorescent dyes of the same kind, each of the detectorsassociated with a quadrant can be equipped with a different colourfilter so that up to four different emission wavelengths can be measuredsimultaneously. Alternatively, a CCD camera can simply be providedbehind the sample. This camera would, however, mix the fluoresent lightof all four channels. This is prevented by positioning a plate (aso-called face plate) consisting of light-conducting fibres having asmall numerical aperture in front of the camera so as to suppresscross-talk between the channels.

What is claimed is:
 1. A light scanning device for exciting and detecting an emission of secondary light by a sample, comprising: a light generating device for generating scanning light in the form of a single light beam, a deflection unit used for effecting a deflection of the scanning light for scanning at least one subarea of the sample, said deflection being variable in at least one direction, an imaging unit for forming an image of the secondary light emanating from the sample, a detection unit for detecting the secondary light, and a division device in the optical path of the scanning light for dividing the single light beam into at least two light beams, each of the light beams having the same spectral qualities as the single beam.
 2. The light scanning device according to claim 1, wherein the division device is a reflecting wedge-shaped beam splitter with first and second surfaces which form an angle with each other and at which the single light beam is reflected, whereby two light beams enclosing the same angle with each other are formed.
 3. The light scanning device according to claim 2, wherein the division device comprises two wedge-shaped bimirrors which are arranged so that the single light beam is reflected at the first reflecting, wedge-shaped beam splitter, whereby two light beams are formed which impinge upon the second reflecting, wedge-shaped beam splitter and which are reflected at this second reflecting beam splitter, whereby four light beams are formed.
 4. The light scanning device according to claim 3, wherein the two reflecting, wedge-shaped beam splitters constitute part of the deflection unit and are connected to adjusting elements for rotating and/or displacing the respective bimirrors for scanning the sample.
 5. The light scanning device according to claim 1, wherein a focussing optics is provided between the division device and the sample for focussing all light beams onto the sample.
 6. The light scanning device according to claim 5, wherein the focussing optics comprises an F/θ lens.
 7. The light scanning device according to claim 1, wherein the detection unit is implemented so that it is suitable for detecting the secondary light in a spatially resolved manner.
 8. The light scanning device according to claim 7, wherein the detection unit is subdivided into fields in correspondence with the number of scanning light beams impinging upon the sample.
 9. The light scanning device according to claim 8, wherein a diaphragm subdivided in correspondence with the fields of the detection unit is arranged in front of said detection unit.
 10. The light scanning device according to claim 1, wherein the imaging unit is arranged so that it is adapted to accept secondary light transmitted by the sample.
 11. The light scanning device according to claim 10, wherein on a side of the sample located opposite a side where the scanning light is incident, light guides arranged to form a bundle and which are subdivided into sub-bundles at their detector-side end according to respective scanning areas of the scanning light beams impinging upon the sample, each of said sub-bundles having associated therewith detectors of their own.
 12. The light scanning device according to claim 11, wherein respective wavelength filters are disposed between the sub-bundles of light guides and the respective detectors.
 13. The light scanning device according to claim 12, wherein the wavelength filters are colour filters for the respective different wavelengths of the secondary light.
 14. The light scanning device according to claim 10, wherein the imaging unit is a plate comprising light-conducting fibres having a small numerical aperture and wherein the detection unit comprises a CCD camera.
 15. The light scanning device according to claim 1, wherein an arrangement for detecting the secondary light is a reflective, non-confocal arrangement.
 16. The light scanning device according to claim 15, wherein the imaging unit comprises a dichroic beam splitter by means of which the optical path of the scanning light can be separated from that of the secondary light emanating from the sample.
 17. The light scanning device according to claim 1, wherein the light generating device used for generating the scanning light in the form of a single beam comprises a laser.
 18. The light scanning device according to claim 1, wherein the light-generating device used for generating the beam comprises a device for spatially filtering the single beam.
 19. The light scanning device according to claim 1, wherein the single light beam and each light beam divided from the single light beam have a specific wavelength.
 20. A light scanning device for exciting and detecting an emission of secondary light by a sample, comprising: a light generating device for generating scanning light in the form of a single light beam, a deflection unit used for effecting a deflection of the scanning light for scanning at least one subarea of the sample, said deflection being variable in at least one direction, an imaging unit for forming an image of the secondary light emanating from the sample, a detection unit for detecting the secondary light, and a division device in the optical path of the scanning light for dividing the single light beam into at least two light beams, the division device being a reflecting wedge-shaped beam splitter with first and second surfaces which form an angle with each other and at which the single light beam is reflected, whereby two light beams enclosing the same angle with each other are formed.
 21. The light scanning device according to claim 20, wherein the division device comprises two wedge-shaped bimirrors which are arranged so that the single light beam is reflected at the first reflecting, wedge-shaped beam splitter, whereby two light beams are formed which impinge upon the second reflecting, wedge-shaped beam splitter and which are reflected at this second reflecting beam splitter, whereby four light beams are formed.
 22. The light scanning device according to claim 21, wherein the two reflecting, wedge-shaped beam splitters constitute part of the deflection unit and are connected to adjusting elements for rotating and/or displacing the respective bimirrors for scanning the sample.
 23. A light scanning device for exciting and detecting an emission of secondary light by a sample, comprising: a light generating device for generating scanning light in the form of a single light beam, a deflection unit used for effecting a deflection of the scanning light for scanning at least one subarea of the sample, said deflection being variable in at least one direction, an imaging unit for forming an image of the secondary light emanating from the sample, a detection unit for detecting the secondary light, a division device in the optical path of the scanning light for dividing the single light beam into at least two light beams, and wherein on a side of the sample located opposite a side where the scanning light is incident, light guides arranged to form a bundle and which are subdivided into sub-bundles at their detector-side end according to respective scanning areas of the scanning light beams impinging upon the sample, each of said sub-bundles having associated therewith detectors of their own.
 24. The light scanning device according to claim 23, wherein respective wavelength filters are disposed between the sub-bundles of light guides and the respective detectors.
 25. The light scanning device according to claim 24, wherein the wavelength filters are colour filters for the respective different wavelengths of the secondary light.
 26. The light scanning device according to claim 23, wherein the imaging unit is a plate comprising light-conducting fibres having a small numerical aperture and wherein the detection unit comprises a CCD camera. 